Fluvastatin Decreases The Expression Of Fibronectin By Inhibiting The Signal Passageway Of Rho-kinase In Hk-2 Cells

Diabetic nephropathy is one of the severe microvascular complications, which is the main reason of end stage renal disease and is the major reason of causing death and mutilation in diabetic patients. Anciently, the research on diabetic nephropathy was outweigh in glomerulus, however, recently research acquires that glomerulus and renal tubule are both disfunction in earlier period of diabetic nephropathy, tubulopathy is significant in the process of diabetic nephropathy. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, or statins, are potent inhibitors of cholesterol synthesis that are extensively used in the treatment of hypercholesterolemia. It is usually assumed that the beneficial effects of statins result from the competitive inhibition of cholesterol biosynthesis. However, studies have demonstrated that statins may also exert additional beneficial effects beyond their cholesterol-lowering properties by preventing the synthesis of isoprenoid intermediates, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which form adducts with a range of intracellular signaling molecules such as Rho, Rac, and Ras. Rho, Rac, and Ras are monomeric G proteins, which function as molecular switches to control many eukaryotic cell functions like antiinflammatory and antiproliferation of renal protection. The Rho-kinase signal pathway has recently received considerable attention for its involvement in a wide variety of pathological states ranging from cardiovascular disease and brain disease to renal pathological changes and blood vessel disease. Rho kinase (ROCK), a down stream effector of RhoA, is a serine/threonine kinase family member, which has some effects in the progressing of diabetic nephropathy. In the current study, our goal is to elucidate the mechanisms by which fluvastatin inhibits the activation of Rho-kinase in responseto high glucose. Once activated, Rho-kinase induced production of fibronecin (FN) in HK-2 cells thus promoting the development of diabetic nephropathy. We, therefore, predict that statins may inhibit HK-2cells synthesis of FN by antagonizing high glucose-induced activation of the Rho-kinase signaling cascade.Aim:1. To observe the effect of high glucose exposure on fibronectin expression in cultured human renal tubular epithelial cells.2. To investigate the role of Rho-kinase signal pathway in this process.3. To investigate the mechanism of fluvastatin in preventing the expression of fibronectin induced by high glucose in HK-2s.4. To explore the association of—LPA the excitomotor of Rho-kinase and isoprenoid intermediates—MVA, GGPP, FPP with Rho-kinase signal pathway and fluvastatin.Methods:1. HK-2s culture and subculture: HK-2s were plated on plastic tissue culture flasks in DMEM (pH 7.4) in normal glucose (NG; 5.5 mM) or HG (25 mM) or NG plus mannitol (19.5 mM). Culture medium was supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, 100g/ml streptomycin. Cells were incubated at 37°C in humidified 5% CO2-95% air. When the HK-2s were cultured in exponential phase of growth, the culture media was blotted and tryptar was used 2. HK-2s subgroup and treatment conditionsWhen HK-2s were grown to 75–85% confluence, washed once with serum-free DMEM, and growth-arrested in serum-free DMEM in NG for 24 h to synchronize the cell growth. After this time period, the cells were subgrouped as follow:①high glucose treated for 0h,6h,12h,24h②media was changed to fresh serum-free media containing NG, HG, or NG plus mannitol in the presence or absence of 1×10-7mol/L, 1×10-6mol/L, 1×10-5mol/L fluvastatin.③normal glucose group,high glucose group,high glucose plus 1μM fluvastatin for 12h and 20h, high glucose plus 1μM fluvastatin for 12h and then plus 1×10-6mol/L LPA for 8h.④high glucose plus 1×10-6mol/L fluvastatin in the presence or absence of MVA(2×10-4mol/L),GGPP(10×10-6mol/L)and FPP(10×10-6mol/L)for 12h.3. The expressions of p-MYPT1( representing activity of Rho-kinase) and FN were detected by Western BlotResult :1. Effect of high glucose on the expression of p-MYPT1 and FN (Western Blot)In a time-dependent manner, treatment of HK-2s with high glucose (6h, 12h, 24h) significantly increased Rho-kinase activity as measured by phosphorylation of MYPT1(p<0.05), one of the downstream targets of Rho-kinase . The level of p-MYPT1 stimulated by high glucose reached the peak at 12h (p<0.01).Compared with 0h control group, exposure to hyperglycemia signifecantly increased the expression of FN in a concentration-dependent manner (p<0.05or p<0.01).2. Effect of fluvatatin on high glucose induced production of p-MYPT1 and FN (Western Blot)Compared with normal glucose group, exposured to hyperglycemia signifecantly increased Rho-kinase activity and the expression of FN in HK-2 cells (p<0.05).Similar concentrations of mannitol did not affect Rho-kinase activity and the level of FN, suggesting that changes in osmolarity were not responsible for hyperglycemia-induced Rho-kinase activation and the expression of FN. Fluvastatin inhibited high glucose-induced p-MYPT1 and FN production in concentration-dependent(1×10-7mol/L, 1×10-6mol/L, 1×10-5mol/L) in culured HK-2s (p<0.05), and 1×10-5mol/L fluvastatin had the obvious inhibitory effect(p<0.01).3. Effect of LPA on the Rho kinase activity and FN accumulation (Western Blot)Compared with normal glucose group, the expressions of p-MYPT1 and FN were signifecantly increased when treated with high glucose(p<0.05), while treated with fluvastatin(12h, 20h ) inhibited the increased Rho kinase activity and the accumulation of FN (p<0.05). However, the productions of p-MYPT1 and FN were increased when LPA was plused(p<0.05).4. The effect of statins is mediated through the inhibition of isoprenoids(Western Blot)Compared with high glucose group, the expressions of p-MYPT1 and FN were signifecantly decreased when treated with 1×10-6mol/L fluvastatin and FPP (p<0.05), compared with 1×10-6mol/L fluvastatin group, the expressions of p-MYPT1 and FN were signifecantly increased when treated with MVA and GGPP (p<0.05).Conclusions:1. High glucose can stimulate the expression of p-MYPT1 and fibronectin.2. Fluvastatin can inhibited high glucose-induced p-MYPT1 and FN production in dose-dependent.3. Fluvastatin can decrease the expression of fibronectin by inhibiting Rho-kinase signaling pathway.4. Rho-kinase may be one of the initiation signals of renal interstitial fibrosis of diabetic nephropathy.